Targeted modification of a chromosomal gene in a living cell is central to the development of gene therapy. To be maximally effective, such targeted modification results in the change of one or more nucleotides in the sequence of a chromosomal gene. Specific examples include conversion of a mutant allele into its wild-type counterpart and inactivation of a deleterious gene by creating a nucleotide sequence specifying premature transcriptional or translational termination, or altered RNA processing.
A serious challenge to the development of effective compositions and methods for targeted modification has been the difficulty in designing modifying agents which are capable of stable interaction with a target sequence, but retain the specificity necessary for targeted modification. For example, certain intercalating agents have a high affinity for DNA, but react non-specifically with numerous different DNA sequences. On the other hand, reagents that are highly specific for a particular nucleotide sequence, such as complementary oligonucleotides, often do not have sufficient affinity for a target sequence to allow efficient targeted modification to proceed on a reasonable time scale.
Several approaches to sequence-specific modification of a target double-stranded nucleotide sequence have been attempted. The use of triplex-forming oligonucleotides with attached modifying groups has been described in WO 94/17092 and WO 96/40711. These reagents are capable of recognizing a target sequence comprising base-paired, double-stranded DNA, and forming a triple-stranded structure that is mediated by a type of base-pairing different than Watson-Crick type base-pairing. Fresco, U.S. Pat. No. 5,422,251. Attachment of a suitable chemical modifying agent to such an oligonucleotide makes it possible to generate a lesion at or near a target sequence in a gene of interest. Subsequent cellular processes related to DNA replication, recombination and/or repair can result in either restoration of the original sequence by repair of the lesion, or mutagenesis, for example by misrepair, resulting in a base change at the site of the lesion. However, formation of triplexes that are sufficiently stable to achieve modification of a target sequence require sequences containing at least about 12 consecutive purine residues on one strand. Consequently, targeting strategies utilizing modified triplex-forming oligonucleotides are restricted to genes having the requisite homopurine runs.
An alternative approach to targeted modification involves the use of modified oligonucleotides having traditional Watson-Crick complementarity to a target sequence, in concert with a recombinase enzyme. The recombinase enzyme facilitates strand invasion at the target sequence by the complementary oligonucleotide, with the formation of a D-loop-type structure. See WO 93/03736 and WO 96/40711. Efficient formation of this structure and hence, efficient modification, requires at least approximately 26 nucleotides of homology between the oligonucleotide and its target sequence, as described in WO 96/40711. In addition, the method depends on either deliberate or fortuitous interaction between the oligonucleotide and a recombinase enzyme, which may be difficult to control.
Thus, a facile method for non-enzymatic targeting of specific sequences in double-stranded DNA that is more broadly applicable than conventional triplex targeting, along with compositions for use in such a method, would greatly enhance the field of gene therapy. Methods and compositions designed to facilitate the interaction of a complementary oligonucleotide with a target sequence have heretofore relied on attaching the oligonucleotide to an agent having non-specific affinity for DNA, such as an intercalating agent, staphylococcal nuclease or short synthetic positively-charged peptides. U.S. Pat. No. 4,835,263; Mouscadet et al. (1994) Biochemistry 33:4187-4196; Corey et al. (1995) Bioconjug. Chem. 6:93-100; and Iyer et al. (1995) J. Biol. Chem. 270:14712-14717. However, these agents possess only a weak general affinity for DNA and thus are not able to localize the oligonucleotide to the vicinity of its target sequence.
Displacement loop (D-loop) formation offers, in principle, no limits on targeting sequence but faces significant thermodynamic and topological issues. Peptide nucleic acids can form D-loop like structures by strand invasion, but only at homopurine runs. The versatility of Watson-Crick sequence targeting might be realized if: (a) D-loop formation could be facilitated and (b) the unstable D-loop could be stabilized.